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Blood, Vol. 109, Issue 2, 566-576, January 15, 2007
Identification of a 2-stage platelet aggregation process mediating shear-dependent thrombus formation
Blood Maxwell et al.
109: 566
Supplemental materials for: Maxwell et al, Vol 109, Issue 2, 566-576
Files in this Data Supplement:
- Supplemental Methods and Materials (PDF, 92.1 KB)
- Video 1. Platelets retain a discoid morphology during translocation on the injured vessel wall in vivo (MOV, 2.67 MB)
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Videomicroscopy showing the earliest stages of platelet interactions with the injured vessel wall in the first minute following vessel injury. Platelets translocating directly on the vessel wall retained a discoid morphology. Drag forces from interactions with other platelets caused stretch and recoil behavior, a characteristic typical of membrane tethers. Scale bar equals 5 µm.
- Video 2. Platelets retain a discoid morphology during translocation on growing thrombi in vivo (MOV, 4.16 MB)
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Mesenteric arterioles of rats were injured via photoactivation of systemically administered rose bengal, leading to the development of small nonocclusive thrombi. Platelet morphology and translocation dynamics were visualized by DIC microscopy. Most platelets interacting with the thrombus retained a discoid morphology and translocated with a sliding motion. Platelets in the superficial layers of forming thrombi were initially loosely tethered to each other and readily detached from the thrombus surface either as individual cells or small clusters of loosely adherent platelets (blue arrow). The vessel wall is outlined in yellow. Scale bar equals 10 µm.
- Video 3. Discoid platelets in the superficial layers of forming thrombi in vivo stretch and recoil under the influence of drag forces (MOV, 4.07 MB)
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Videomicroscopy showing platelets within the superficial layers of a forming thrombus. Many discoid platelets displayed free movement, undergoing stretch and recoil behavior, and even free rotation. The schematic shows an individual platelet (purple outline) tethered to a growing thrombus (blue outline), which extends and recoils as drag forces are exerted from interactions with other platelets (yellow outline) and then eventually detaches. Scale bar equals 3 µm.
- Video 4. Reversible aggregation of discoid platelets during translocation on VWF in vitro (MOV, 3.91 MB)
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Whole blood was perfused through VWF-coated microcapillary tubes (50 µg/mL) at 1800 s–1. The video shown was recorded during the period of 90 to 120 seconds of perfusion, showing recruitment of discoid platelets into aggregates (blue circles). The higher-magnification section highlights the central partially activated platelet (blue arrow), which acts as a nucleus for recruitment of additional platelets, typically adhering with transient, reversible interactions. Scale bar equals 10 µm.
- Video 5. Increasing matrix reactivity promotes aggregate formation (MOV, 5.52 MB)
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Washed platelets reconstituted with RBCs at a concentration of 300 × 106/mL were perfused through microcapillary tubes coated with a mixture of VWF (10 µg/mL) and fibrinogen (0, 10, 20, and 50 µg/mL) at 1800 s–1. The video shows clips of 5-second duration at the 30-, 60-, 90-, and 120-second time points. Unstable aggregates are circled in blue, and the conversion to stable aggregates is indicated by subtitles. The videos shown are representative of at least 4 independent experiments.
- Video 6. Formation of stable aggregates is promoted by increasing platelet density under in vitro flow conditions (MOV, 4.43 MB)
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Washed platelets were reconstituted with RBCs to a final concentration of 5 × 106/mL, 20 × 106/mL, 150 × 106/mL, or 800 × 106/mL and perfused through microcapillary tubes coated with a mixture of VWF (10 µg/mL) and fibrinogen (50 µg/mL) at 1800 s–1. The video shows clips of 5-second duration at the 30-, 60-, 90-, and 120-second time points. Formation of unstable and stable aggregates is indicated by subtitles. The videos shown are representative of at least 3 independent experiments.
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